Fumiko Ozawa

Novel Members of the Vesl/Homer Family of PDZ Proteins That Bind Metabotropic Glutamate Receptors

Vesl-1S (186 amino acids, also called Homer) is a protein containing EVH1- and PDZ-like domains whose expression in the hippocampus is regulated during long term potentiation (LTP), one form of synaptic plasticity thought to underlie memory formation (Kato, A., Ozawa, F., Saitoh, Y., Hirai, K., and Inokuchi, K. (1997) FEBS Lett. 412, 183–189; Brakeman, P. R., Lanahan, A. A., O’Brien, R., Roche, K., Barnes, C. A., Huganir, R. L., and Worley, P. F. (1997)Nature 386, 284–288). Here we report additional members of the Vesl/Homer family of proteins, Vesl-1L and Vesl-2. Vesl-1L (366 amino acids), a splicing variant of Vesl-1S, shares N-terminal 175 amino acids with Vesl-1S and contains additional amino acids at the C terminus. Vesl-2 (354 amino acids) was highly related to Vesl-1L in that both contain EVH1- and PDZ-like domains at the N terminus (86% conservation) and an MCC (mutated in colorectal cancer)-like domain and a leucine zipper at the C terminus. In contrast to vesl-1S, we observed no changes in the levels of vesl-1L andvesl-2 mRNAs during dentate gyrus LTP. All these proteins interacted with metabotropic glutamate receptors (mGluR1 and mGluR5) as well as several hippocampal proteins in vitro. Vesl-1L and Vesl-2, but not Vesl-1S, interacted with each other through the C-terminal portion that was absent in Vesl-1S. Vesl-1L and Vesl-2 may mediate clustering of mGluRs at synaptic junctions. We propose that Vesl-1S may be involved in the structural changes that occur at metabotropic glutamatergic synapses during the maintenance phase of LTP by modulating the redistribution of synaptic components.

vesl, a gene encoding VASP/Ena family related protein, is upregulated during seizure, long-term potentiation and synaptogenesis.

We have isolated a novel cDNA, vesl, that was induced during convulsive seizure in the rat hippocampus. The vesl gene encodes a protein of 186 amino acids that has significant homology to the EVH1 domain of the VASP/Ena family of proteins implicated in the control of microfilament dynamics. The expression of vesl mRNA was induced in the granule cell layer during persistent long‐term potentiation (LTP) of the dentate gyrus in an NMDA receptor‐dependent manner. Furthermore, vesl mRNA was expressed at a high level during hippocampal synaptogenesis. We suggest that the Vesl protein may be involved in the structural changes that occur at synapses during long‐lasting neuronal plasticity and development.

Differential functional interaction of two Vesl/Homer protein isoforms with ryanodine receptor type 1: a novel mechanism for control of intracellular calcium signaling.

Vesl/Homer proteins physically link proteins that mediate cellular signaling [Curr. Opin. Neurobiol. 10 (2000) 370; Trends Neurosci. 23 (2000) 80; J. Cell Sci. 113 (2000) 1851] and thereby influence cellular function [Nat. Neurosci. 4 (2001) 499; Nature 411 (2001) 962]. A previous study reported that Vesl-1L/Homer-1c (V-1L) controls the gain of the intracellular calcium activated calcium channel ryanodine receptor type 1 (RyR1) channel [J. Biol Chem. 277 (2002) 44722]. Here, we show that the function of RyR1 is differentially regulated by two isoforms of Vesl-1/Homer-1, V-1L and Vesl-1S/Homer-1a (V-1S). V-1L increases the activity of RyR1 while important regulatory functions and pharmacological characteristics are preserved. V-1S alone had no effect on RyR1, even though, like V-1L, it is directly bound to the channel. However, V-1S dose-dependently decreased the effects of V-1L on RyR1, providing a novel mechanism for the regulation of intracellular calcium channel activity and calcium homeostasis by changing expression levels of Vesl/Homer proteins.

Increase in activin βA mRNA in rat hippocampus during long-term potentiation

We have used mRNA differential display to isolate genes that are induced by neural activity in rat hippocampus. One of these encodes activin βA subunit. Convulsive seizure caused by kainate significantly induced the expression of activin βA mRNA. Furthermore high frequency stimulation (HFS) of perforant pathway, which produced a persistent long‐term potentiation (LTP) (> 10 h), caused a marked increase at 3 h in the level of activin βA mRNA at the dentate gyrus of urethane‐anesthetized rat. The increase was NMDA receptor‐dependent. By contrast the level of inhibin α mRNA was not changed following the induction of LTP. The results suggest a role for activin in maintenance of neural plasticity in the adult brain.

Vesl/Homer proteins regulate ryanodine receptor type 2 function and intracellular calcium signaling.

Cellular signaling proteins such as metabotropic glutamate receptors, Shank, and different types of ion channels are physically linked by Vesl (VASP/Ena-related gene up-regulated during seizure and LTP)/Homer proteins [Curr. Opin. Neurobiol. 10 (2000) 370; Trends Neurosci. 23 (2000) 80; J. Cell Sci. 113 (2000) 1851]. Vesl/Homer proteins have also been implicated in differentiation and physiological adaptation processes [Nat. Neurosci. 4 (2001) 499; Nature 411 (2001) 962; Biochem. Biophys. Res. Commun. 279 (2000) 348]. Here we provide evidence that a Vesl/Homer subtype, Vesl-1L/Homer-1c (V-1L), reduces the function of the intracellular calcium channel ryanodine receptor type 2 (RyR2). In contrast, Vesl-1S/Homer-1a (V-1S) had no effect on RyR2 function but reversed the effects of V-1L. In live cells, in calcium release studies and in single-channel electrophysiological recordings of RyR2, V-1L reduced RyR2 activity. Important physiological functions and pharmacological properties of RyR2 are preserved in the presence of V-1L. Our findings demonstrate that a protein-protein interaction between V-1L and RyR2 is not only necessary for organizing the structure of intracellular calcium signaling proteins [Curr. Opin. Neurobiol. 10 (2000) 370; Trends Neurosci. 23(2000)80; J. Cell Sci. 113 (2000) 1851; Nat Neurosci. 4 (2001) 499; Nature 411 (2001) 962; Biochem. Biophys. Res. Commun. 279 (2000) 348; Nature 386 (1997) 284], but that V-1L also directly regulates RyR2 channel activity by changing its biophysical properties. Thereby it may control cellular calcium homeostasis. These observations suggest a novel mechanism for the regulation of RyR2 and calcium-dependent cellular functions.

Expression of β-nerve growth factor mRNA in rat glioma cells and astrocytes from rat brain

A 50‐base synthetic oligodeoxynucleotide complementary to a portion of mouse nerve growth factor (NGF) mRNA was used as a probe for analysis of the expression of NGF gene. Northern blot analysis showed the presence of a major 1.3 kb transcript, which was identical in size to mouse NGF mRNA, in both C6Bu1 cells and rat astrocytes cultured from newborn rat brain. Further, the rearrangement of DNA sequence in and around the NGF gene locus of C6Bu1 cells was not detected by Southern blot analysis. These results indicate the expression of NGF mRNA in both C6Bu1 cells and astrocytes from rat brain, suggesting that astrocytes may produce NGF protein in the rat brain, especially in developing rat brain.

Input-specific spine entry of soma-derived Vesl-1S protein conforms to synaptic tagging.

Synaptic Tag Tagged Input-dependent synaptic plasticity is critical for the reproducible activation of a specific neuronal assembly encoding a particular memory. The synaptic tagging hypothesis, which suggests how input specificity is maintained in late-phase synaptic plasticity, attempts to explain the persistence of long-term memory. However, it has been difficult to identify proteins that behave as the hypothesis predicts. Okada et al. (p. 904) investigated if the regulated spine entry of a late-phase-related somatically synthesized plasticity-related protein, Vesl-1S, works as a synaptic tag. Vesl-1S protein was carried from the soma to every dendrite and recruited into spines by synaptic activation in an input-specific manner. Spine entry was protein-synthesis independent, was NMDA receptor dependent, and had a persistent lifetime of activation. These results provide long-sought evidence for the input-specific capturing of a plasticity-related protein as postulated by the synaptic tagging hypothesis. The protein Vesl-1S fulfills the synaptic tagging hypothesis for the maintenance of input-specific action of neuronal networks. Late-phase synaptic plasticity depends on the synthesis of new proteins that must function only in the activated synapses. The synaptic tag hypothesis requires input-specific functioning of these proteins after undirected transport. Confirmation of this hypothesis requires specification of a biochemical tagging activity and an example protein that behaves as the hypothesis predicts. We found that in rat neurons, soma-derived Vesl-1S (Homer-1a) protein, a late-phase plasticity-related synaptic protein, prevailed in every dendrite and did not enter spines. N-methyl-d-aspartate receptor activation triggered input-specific spine entry of Vesl-1S proteins, which met many criteria for synaptic tagging. These results suggest that Vesl-1S supports the hypothesis and that the activity-dependent regulation of spine entry functions as a synaptic tag.

Regulated expression of an actin-associated protein, synaptopodin, during long-term potentiation

We report NMDA receptor‐dependent expression of synaptopodin mRNA in the dentate granule cells of the hippocampus following induction of long‐term potentiation (LTP) in vivo. Synaptopodin did not belong to immediate‐early genes, as de novo protein synthesis was required for the induction of synaptopodin gene transcription. An increased level of synaptopodin mRNA was observed at 75 min and 3.5 h after the onset of LTP. Importantly, there was correlation between the induction of mRNA expression and the persistence of LTP. Synaptopodin immunoreactivity was elevated specifically in synaptic layers, middle and outer molecular layers of dentate gyrus where LTP was induced. As synaptopodin is an actin‐associated protein present in spine neck and implicated in the modulation of cell morphology, our results suggest that synaptopodin, by regulating the dynamics of the actin cytoskeleton, contributes to the morphological change in spine shape considered to be important for the maintenance of synaptic plasticity.

A facilitatory effect on the induction of long-term potentiation in vivo by chronic administration of antisense oligodeoxynucleotides against catalytic subunits of calcineurin

A rise in Ca2+ concentration at postsynaptic sites provides an initial step in inducing both the long-term potentiation (LTP) and long-term depression (LTD) in the CA1 region of the hippocampus. LTP induction requires the activation of Ca(2+)-sensitive protein kinases following the rise in Ca2+. By contrast, the activity of protein phosphatase(s) appears to be critical to induce LTD. Here we demonstrate that inhibition of the synthesis of calcineurin A alpha and A beta, catalytic subunits of Ca2+/calmodulin- (CaM) dependent protein phosphatase, reduces the threshold of induction for commissural-CA1 LTP in anesthetized rats. In rats administered antisense oligodeoxynucleotides (ODNs) against calcineurin A alpha and A beta intraventricularly for 7 days, a brief tetanic stimulation to the CA3 region, which in the control case was below threshold for the induction of LTP, now produced a long-lasting increase in both the EPSP slope and the amplitude of population spike recorded from the commissural-CA1 pathway. Western blot analysis of calcineurin showed that the threshold reduction was accompanied by a selective decrease in the protein levels in the hippocampus. Thus our study provides direct evidence that calcineurin per se has an antagonizing role in LTP induction. Complementary experiments with the selective calcineurin inhibitor, FK506, also showed the reduction of LTP threshold in a dose-dependent manner. These results, together with previous studies, support the hypothesis that the quantitative phosphorylation level of critical intracellular proteins determines whether the synaptic efficacy will increase or decrease after the activity-dependent rise in postsynaptic Ca2+.

Biologically active human and mouse nerve growth factors secreted by the yeast Saccharomyces cerevisiae

Nerve growth factor (NGF) is a trophic agent that is essential for the development and survival of sympathetic and sensory nerves. A chemically-synthesized DNA fragment encoding human NGF (hNGF) and a cDNA encoding mouse NGF (mNGF) were engineered for expression in the yeast, Saccharomyces cerevisiae. Expression and secretion of hNGF and mNGF was attempted under the direction of the yeast PGK promoter and with various leader sequences. Among the leader sequences tested, that of the yeast α-factor successfully directed secretion of both hNGF and mNGF that were correctly processed. The content of the recombinant NGF (reNGF) in the culture supernatant was estimated to be 1 μg/ml. The yeast-produced reNGF was able to bind to NGF receptors in rat pheochromocytoma (PC12) cells as efficiently as the standard mNGF, and partially purified reNGF could induce neurite outgrowth of PC12 cells. Thus, we have demonstrated that biologically active human and mouse reNGF can be produced in yeast cells.